Epstein-Barr Virus (EBV) establishes long-term latent infections that can drive formation of diverse lymphomas and carcinomas. EBV-associated tumors harbor latent viral genomes that persist as multicopy, chromatin- associated episomes that express a limited subset of viral genes. The viral encoded protein EBNA1 is the only viral protein consistently expressed in all EBV-associated tumors. EBNA1 is a multifunctional, sequence- specific DNA-binding protein that regulates viral DNA replication, episome maintenance through mitosis, metaphase chromosome tethering, chromatin structure, and transcription. EBNA1 is also required for host-cell survival. The molecular mechanisms for each of these functions are only partly understood, and a more complete understanding is necessary for development of effective strategies to treat EBV latent infection and carcinogenesis. During the previous funding cycles, we found that EBNA1 DNA-binding domain has complex recombination-like functions at the viral origin of replication (OriP) and episome maintenance element. We have used X-ray crystallography to solve new structures of EBNA1 revealing novel oligomeric structures and interfaces important for DNA replication and episome maintenance. We have used functional genomic approaches, including CHIP-Seq and RNA-Seq to identify EBNA1-cellular chromosome binding sites and candidate EBNA1-regulated cellular genes implicated in host cell growth and survival pathways. We have characterized a naturally occurring variant of the EBNA1 DNA binding domain that confers distinct biochemical and functional properties correlating with EBV carcinogenic risk. We now propose to advance these studies to better understand the role of EBNA1 in viral episome maintenance and host-cell survival during latent infection. We propose to test the overarching hypothesis that EBNA1 utilizes previously unrecognized mechanisms to coordinate multiple aspects of viral genome persistence with host-cell fitness. We will test the specific hypotheses that (1) EBNA1 promotes recombination-dependent replication and mitotically stable episome maintenance at OriP through unique structural features in its DNA binding domain, (2) EBNA1 provides host- cell survival advantage by modulating chromosome architecture and epigenetic control of cellular oncogenic factors, and (3) EBNA1 exists in variant forms that may have different functional properties and oncogenic potential. More detailed understanding of EBNA1 is further justified by its potential value as a target for therapeutic intervention in cancers and other diseases associated with EBV latent infection.